The Whitley Internal HEPA filtration system bacteriological testing

Transcription

1 The Whitley Internal HEPA filtration system bacteriological testing Andrew Pridmore July 2014 Introduction As described in Technical Note HE03, Whitley Workstations equipped with our Whitley Internal HEPA Filtration System achieve particle counts in the internal atmosphere that exceed the requirements of ISO Class 3. To our knowledge, no other manufacturers of positive pressure modified atmosphere workstations can approach anything like compliance with the stringent standards we attain. Please review other DWS Technical Notes in this series for a complete understanding of the superiority of the Whitley Internal HEPA Filtration System. Due to the types of the work performed by users of our hypoxia workstations and anaerobic workstations, we consider it important to augment the particle count data with specific proof that the HEPA filtration system is capable of removing bacteria from the atmosphere. Therefore, we conducted further laboratory tests to challenge the atmosphere with high concentrations of aerosolized bacterial cells and spores and to quantify their removal Don Whitley Scientific Limited / All rights reserved Page 1 of 6

2 Test Method An H35 HEPA and an H45 HEPA hypoxystation were tested. In addition, a standard H35 hypoxystation (no HEPA) was tested to provide comparative data. Bacterial strains used for the test were the non sporeforming aerobe Kocuria rhizophila and the sporeforming anaerobe Clostridium beijerinckii (both with low pathogenic potential). To produce a suspension of vegetative cells, K. rhizophila was subcultured aerobically on Tryptone Soy Agar (TSA) at 37 C for 2 days. Cells were harvested into sterile Maximum Recovery Diluent. To produce a spore suspension, C. beijerinckii was cultured anaerobically in ml volumes of Bryant and Burkey medium at 37 C for 10 days. The two liquid cultures were heated at 80 C for 10 minutes, centrifuged to pellet the spores and cell debris, washed 3 times in sterile 0.85% NaCl and the combined material from both cultures was resuspended in 100 ml of sterile 0.85% NaCl to concentrate the spores. Viable counts (determined by plating) were > cfu/ml for K. rhizophila cell suspension and > cfu/ml for C. beijerinckii spore suspension. Before testing, each workstation was allowed to stabilize at 37 C and 75% relative humidity and was operated continuously throughout the test so that normal atmospheric circulation occurred. A 55 ml volume of K. rhizophila cell suspension or C. beijerinckii spore suspension was added to a 6 jet NSF Collison nebulizer (BGI Inc; and the fully assembled nebulizer was weighed Don Whitley Scientific Limited / All rights reserved Page 2 of 6

3 The nebulizer was transferred into the workstation, placed on the upper shelf with the outlet facing the front of the chamber and connected to compressed air at 20 psi pressure and 12 litres per minute flow rate. Nebulization was performed for 5 minutes, after which the nebulizer was weighed again to determine the dispensed volume. Sampling of the internal workstation atmosphere was conducted using an AES Sampl air Lite (biomérieux). Samples of 100 litres were collected by impaction onto a 90 mm petri dish of TSA (K. rhizophila experiments) or Fastidious Anaerobe Agar (FAA; C. beijerinckii experiments). The atmosphere was sampled during the final 1 minute of nebulization, immediately after completion of nebulization and at timed intervals during the subsequent 30 minutes Don Whitley Scientific Limited / All rights reserved Page 3 of 6

4 Results Bacterial colony count data obtained from the air sampler plates are tabulated below (note that the upper detection limit for the air sampler is 258 colonies). The standard H35 workstation allowed K. rhizophila to persist, without a measurable reduction in viable count, for at least 30 minutes after nebulization (Table 1). An additional sample after 4 hours also revealed high numbers of the organism (not shown in the table). In contrast, K. rhizophila count in the atmosphere of the H35 HEPA hypoxystation 2 minutes after nebulization was reduced more than 100-fold in comparison with the standard H35 and the organism could not be recovered from the H35 HEPA after 5 minutes (Table 2). Results obtained in the H45 HEPA were similar (Table 3). On the basis of these results, the performance of H35 / H45 HEPA hypoxystations exceed the requirement stipulated in ISO that the appropriate class of atmospheric cleanliness should be regained within 20 minutes following any disruption. This is in agreement with the particle count data reported in Technical Note HE03. To test the capacity of the HEPA filter to remove bacterial spores, nebulization experiments were conducted in the H35 HEPA using C. beijerinckii spores. Sampling of the internal atmosphere after nebulization of the spore suspension produced a small colony count immediately after the nebulizer was stopped, then no recovery of the organism after 2 minutes. Thus, the HEPA filter was also highly effective in the removal of spores from the atmosphere. The shorter time required to achieve zero counts, in comparison with the K. rhizophila results, was probably attributable to the lower viable count attained in the spore suspension. Conclusions Our experiments have demonstrated that the HEPA filtration system in Whitley workstations achieves a rapid and substantial reduction in bacterial contamination of the atmosphere. It should be noted that these results are equally applicable to the equivalent models of anaerobic workstation (A35 HEPA) because their HEPA filtration systems are identical to those in the equivalent model of H workstation Don Whitley Scientific Limited / All rights reserved Page 4 of 6

5 2014 Don Whitley Scientific Limited / All rights reserved Page 5 of 6

6 This work was presented at Anaerobe 2014, Chicago, USA. A copy of the poster is available to download from This series of technical papers is commissioned by Don Whitley Scientific Limited to provide customers with technical, scientific and product assistance. These papers are commissioned from a variety of sources, including our own internal contract laboratories, but usually with the kind co-operation of our customers. These papers are published with the best of intentions and are based on the latest information available to us at the time of publication. Don Whitley Scientific cannot assume responsibility for individual use or application of the information contained in this series of papers, however, we encourage readers to contact us with their experiences on the topics covered Don Whitley Scientific Limited / All rights reserved Page 6 of 6